The broad and long-term objectives of the application are to understand the molecular mechanisms of neuronal network formation. During development of neuronal networks, axons must follow correct pathways to reach and recognize their correct targets. Initial development of these neuronal projections is believed to be established through the interaction between multiple guidance cues derived from the pathways and targets, and their cognate receptors expressed on growing axons. The proposed studies focus on the EphB6 receptor, a unique member of the Eph family of receptor tyrosine kinases. The Eph family is the largest family of receptor tyrosine kinases, and has been shown to act as key guidance molecules in neuronal network formation. In most cases, ligand binding to full-length Eph receptors mediates repulsive signals, which result in growth cone collapse and axon retraction. However, unlike the other members of the family, EphB6 contains multiple substitutions of conserved amino acid residues in its tyrosine kinase domain and is catalytically inactive, suggesting that EphB6 has unique functions among the Eph family members. We hypothesize that EphB6 mediates adhesive and/or attractive signals, rather than repulsive signals, and acts as an axon guidance receptor. Our preliminary analyses of the central olfactory system of EphB6 null mice support this hypothesis. In this proposal, we will further test this hypothesis in the following studies. First, expression and binding patterns of EphB6 and its cognate ligand, ephrin-B2, will be determined in the developing murine nervous system, by RNA in situ hybridization and affinity probe in situ. Second, functions of the EphB6 receptor in neuronal network formation will be examined both in vivo and in vitro. Based on our preliminary results, we will focus initially on the central olfactory projection. In vivo functions of EphB6 will be examined in axon tracing experiments using EphB6 null mice. In vitro, effects of ephrin-B2 on olfactory bulb axons from wild type and EphB6 null mice will be tested in the membrane stripe assay and axon outgrowth assay. Third, functions and signaling mechanisms of EphB6 in cell adhesion will be analyzed in cell adhesion assays using cell lines that stably express variant forms of EphB6 receptors. Results from the application will provide novel insights into the functions of "defective" receptor tyrosine kinases in neuronal network formation. In addition, since many congenital disorders affect the central nervous system including the olfactory system, they will also provide essential information on the pathogenesis of such disorders.